Amino acid sequence of the BSC-1 cell growth inhibitor (polyergin) deduced from the nucleotide sequence of the cDNA.

The complete amino acid sequence of the BSC-1 cell growth inhibitor, including its precursor polypeptide, is reported. The sequence was deduced from the nucleotide sequence of the cDNA. The N-terminal amino acid sequence of the mature bioactive BSC-1 cell growth inhibitor is identical with the N-terminal sequences of the factors that have been called type beta 2 transforming growth factor and cartilage-inducing factor B, suggesting that these are identical. The complete amino acid sequence of the mature BSC-1 cell growth inhibitor differs from that of human type beta transforming growth factor in 32 of the 112 amino acids. Polyergin is proposed as the name for the BSC-1 cell growth inhibitor.

Insulin-like synergistic stimulation of DNA synthesis in Swiss 3T3 cells by the BSC-1 cell-derived growth inhibitor related to transforming growth factor type beta.

A cell growth inhibitor (GI), purified from BSC-1 cell-conditioned medium, has little if any effect on DNA synthesis when added alone to monolayer cultures of quiescent Swiss mouse 3T3 cells in serum-free medium. However, the inhibitor, which is closely related to transforming growth factor type beta (TGF-beta), exhibits a pronounced synergistic stimulation of DNA synthesis in combination with certain peptide (bombesin, vasopressin) or polypeptide (platelet-derived growth factor) mitogens. A similar synergistic response has been demonstrated for TGF-beta purified from human platelets. In the presence of 3 nM bombesin, a half-maximal stimulation of DNA synthesis was obtained at a GI concentration of approximately 60 pg/ml, with a maximal response at approximately 600 pg/ml. The synergistic interactions demonstrated by GI or TGF-beta in stimulating Swiss 3T3 cells closely resemble those previously shown for insulin, and we have observed that GI does not synergize with insulin to stimulate DNA synthesis in these cells. Like insulin, and in contrast to bombesin, vasopressin, and platelet-derived growth factor, GI does not activate cellular inositolphospholipid hydrolysis, calcium mobilization, or cross-regulation of epidermal growth factor receptor affinity. These results raise the possibility that the biochemical pathways activated by GI/TGF-beta and insulin converge at a post-receptor stage.

BSC-1 growth inhibitor transforms a mitogenic stimulus into a hypertrophic stimulus for renal proximal tubular cells: relationship to Na+/H+ antiport activity.

Renal hypertrophy is characterized by an increase in cell size and protein content with minimal hyperplasia. The mechanisms of control of this pattern of cell growth have not been determined. The present studies examined whether the growth inhibitor elaborated by BSC-1 kidney epithelial cells (GI), which has nearly identical biological properties to transforming growth factor beta (TGF-beta), could transform a mitogenic stimulus into a hypertrophic stimulus for rabbit renal proximal tubular cells in primary culture. Insulin (10 micrograms/ml) plus hydrocortisone (50 nM) increased the amount of protein per cell, cell volume, and [3H]thymidine incorporation at 24 and 48 hr in these cells. GI/TGF-beta (10 units/ml) led to a minimal stimulation of [3H]thymidine incorporation. When added together with insulin plus hydrocortisone, GI/TGF-beta inhibited the stimulatory effect of these mitogens on [3H]thymidine incorporation but did not block the increase in protein per cell and cell volume--i.e., the cells underwent hypertrophy. The fact that this pattern persisted for 48 hr indicated that GI/TGF-beta exerted a prolonged inhibitory effect on mitogenic-stimulated DNA synthesis rather than delaying its onset. Amiloride-sensitive Na+ uptake (indicative of Na+/H+ antiport activity) correlated with protein per cell and cell volume rather than with DNA synthesis. P60 gel chromatographic fractionation of conditioned medium harvested from proximal tubular cells yielded a fraction that inhibited [3H]thymidine incorporation in BSC-1 cells and CCL 64 cells; the relative inhibitory activity on these cell lines and the chromatographic behavior were similar to those observed with GI/TGF-beta. These studies indicate that the control of cell size may be regulated by autocrine mechanisms mediated by the elaboration of growth inhibitory factors that alter the pattern of the growth response to mitogens.

A growth regulatory factor that can both inhibit and stimulate growth.

A growth inhibitor that is produced by BSC-1 cells (African green monkey kidney epithelial cells) has been isolated from conditioned medium. It has been purified by gel chromatography and high performance liquid chromatography. It appears to be a protein with a relative molecular mass (Mr) of 24 000. It is extremely active as a growth inhibitor with some cells, but not with others. Approximately 50% inhibition of thymidine incorporation is observed with CCL64 cells at 0.05 ng/ml and with BSC-1 cells at 1 ng/ml. The growth inhibitor induces BSC-1 cells to synthesize and secrete a glycoprotein of approximately 48 000 Mr. It inhibits Na+ accumulation in BSC-1 cells. Recently, in collaboration with R. F. Tucker, G.D. Shipley and H. L. Moses (Mayo Foundation & Medical School), we have found that the growth inhibitor is very similar to and may be identical with transforming growth factor beta (TGF-beta). Our growth inhibitor stimulates colony formation in soft agar by AKR-2B cells, and it competes with TGF-beta in binding to cell surface receptors. TGF-beta, from human platelets, is extremely active as an inhibitor of thymidine incorporation by BSC-1 cells and CCL64 cells. The growth inhibitor/TGF-beta can, therefore, stimulate or inhibit growth, depending on the cells and the growth conditions.

Growth inhibitor from BSC-1 cells closely related to platelet type beta transforming growth factor.

Purified growth inhibitor from BSC-1 cells and type beta transforming growth factor from human platelets are shown to have nearly identical biological activity and to compete for binding to the same cell membrane receptor. These findings suggest that the growth inhibitor and the type beta transforming growth factor are similar molecules. The data also show that the same purified polypeptide can either stimulate or inhibit cell proliferation depending on the experimental conditions.

Cell growth and net Na+ flux are inhibited by a protein produced by kidney epithelial cells in culture.

Proliferation of confluent kidney epithelial cell cultures (BSC-1 line) is inhibited by a protein (Mr approximately equal to 24,000) that is secreted by the cells. The mechanism of action of this growth inhibitor was sought by studying its effect on net Na+ flux because increased availability of Na+ in the culture medium had been shown to stimulate cell growth. The increase in cell Na+ content observed during stimulation of the growth after a medium change was attenuated in the presence of the purified inhibitor. Inhibition of both cell Na+ accumulation and growth in the presence of the protein was reversed completely by addition of NaCl to the medium. These results suggest that control of net Na+ flux and growth in kidney epithelial cells could be mediated, at least in part, by a secreted cellular protein.

Rapid selective effects by a growth inhibitor and epidermal growth factor on the incorporation of [35S]methionine into proteins secreted by African green monkey (BSC-1) cells.

Confluent African green monkey kidney (BSC-1) cells secrete a protein (Mr approximately equal to 24,000) that inhibits DNA synthesis and growth of the same cells. Using [35S]methionine to metabolically label proteins, we have found that this growth inhibitor selectively induces the BSC-1 cells to synthesize and secrete another protein with a relative Mr of 48,000 on NaDodSO4/polyacrylamide gels. We have called this protein "inhibitor-inducible protein" (IIP48). The maximal increase in rate of labeling of IIP48 due to treatment with the growth inhibitor averages 12-fold over the control. IIP48 is an N-glycosidically linked glycoprotein, and it is not a major intracellular protein. This protein is maximally induced within 4 to 6 hr of adding the growth inhibitor to the cells. This is an early response of these cells to the growth inhibitor and may represent a primary response to the growth inhibitor. Epidermal growth factor (EGF) increases the rate of labeling of three other secreted proteins (MrS 28,000, 59,000, and 61,000), which we have called "mitogen-inducible proteins" (MIP28, MIP59, and MIP61). The specific effects of both EGF and the growth inhibitor on the secreted levels of these proteins are inhibited if actinomycin D is added with the growth effectors. Thus, RNA synthesis appears necessary for the inductions. EGF and the growth inhibitor induce these secreted proteins by independent and noninteracting pathways.

Activity of a kidney epithelial cell growth inhibitor on lung and mammary cells.

A kidney epithelial cell growth inhibitor, isolated from BSC-1 cell-conditioned medium, has been found to be active on certain lung and mammary gland cell lines in culture. The most responsive cell observed thus far is the CCL64 mink lung cell line. With CCL64 cells, 60% inhibition of [3H]thymidine incorporation is observed at a 0.1 nanogram/ml concentration of the growth inhibitor, and approximately 95% inhibition at 1 nanogram/ml. A human mammary tumor cell line, Hs578T, shows 75% inhibition of [3H]thymidine incorporation, in cell culture. Preliminary studies indicate that injection of the kidney epithelial cell growth inhibitor in vivo into human mammary carcinomas growing in nude mice inhibits [3H]thymidine incorporation in the tumors.

Interactions between the growth effects of a kidney epithelial cell growth inhibitor and extracellular concentrations of cyclic nucleotides.

The effect of dibutyryl cyclic nucleotides, dbcAMP and dbcGMP, on the growth of BSC-1 cells has been studied in the presence and absence of kidney epithelial cell growth inhibitor. The growth response of the cells was dependent on concentrations of the dibutyryl cyclic nucleotides and on the presence or absence of serum in the medium. In the presence of serum, a high concentration (10(-3) M) of dbcGMP largely overcame the action of the kidney epithelial cell growth inhibitor. In the absence of serum, a high concentration (10(-3) M) of dbcAMP increased growth inhibition observed with the growth inhibitor. In the presence of serum, low concentrations of dbcAMP were growth stimulatory and partially overcame the action of the growth inhibitor.

Purification of kidney epithelial cell growth inhibitors.

Two high molecular weight growth inhibitors have been isolated from the culture medium of BSC-1 cells, epithelial cells of African green monkey kidney. The purified kidney epithelial cell growth inhibitors, at ng/ml concentrations, reversibly arrest the growth of BSC-1 cells in the G1 phase of the cell cycle. Their action is selective; they are most active on BSC-1 cells, are less active as inhibitors of the growth of rat lung and human breast epithelial cells, and do not inhibit the growth of 3T3 mouse embryo fibroblasts ad human skin fibroblasts in culture. Their growth inhibitory action on BSC-1 cell cultures is counteracted by epidermal growth factor or calf serum.

Control of animal cell proliferation.

Present understanding of the control of animal cell proliferation is summarized briefly. Major gaps in present knowledge are listed. Models of growth control are discussed.

Binding, internalization, and degradation of epidermal growth factor by balb 3T3 and BP3T3 cells: relationship to cell density and the stimulation of cell proliferation.

Epidermal growth factor (EGF) stimulates the growth of both benzo[a]pyrene-transformed Balb 3T3 cells (BP3T3) and untransformed Balb 3T3 cells. We describe here the binding, internalization, and degradation of [125I]-EGF by BP3T3 cells and 3T3 cells. Binding of [125I]-EGF reaches a maximum after 45 to 90 minutes incubation at 37 degrees C. In both BP3T3 and 3T3 cells the extent of EGF binding required to stimulate DNA synthesis is density dependent; sparse cultures require a 15-30% occupancy to elicit a maximal response whereas dense cultures require a 70-85% occupancy. At physiological concentrations the total binding of [125I]-EGF to 3T3 cells is higher than to BP3T3 cells, and this difference increases at higher cell densities. The rate of degradation of [125I]-EGF is directly proportional to the total [125I]-EGF binding in each cell type. This supports the hypothesis that one cause of the diminished serum requirement of BP3T3 cells is a reduced rate of utilization of serum growth factors.

Epidermal growth factor and the control of proliferation of Balb 3T3 and benzo[a]pyrene-transformed Balb 3T3 cells.

Benzo[a]pyrene-transformed Balb 3T3 cells (BP3T3) exhibit "normal" growth controls at low concentrations of serum. Epidermal growth factor (EGF) stimulates DNA synthesis and cell division in both Balb 3T3 and BP3T3 cells at physiological concentrations. The growth response of BP3T3 cells to EGF is qualitatively the same as that of 3T3 cells, however, the transformed cells have a lower quantitative requirement. Both 3T3 and BP3T3 cells show a density-dependent response to EGF, but the shift in the dose response curve for BP3T3 cells at high cell density is smaller than that seen for 3T3 cells. One cause of the restricted growth of 3T3 cells at high cell density compared with BP3T3 cells is the increased concentration of growth factor needed for stimulation of 3T3 cells at higher cell densities. A lower rate of depletion of other growth factory by BP3T3 cells may also explain the smaller effect of cell density on the EGF response of these cells.

Regulation of growth of fibroblasts.

Present knowledge on regulation of fibroblast growth is based on in vitro culture of fibroblasts from different sources. The research has focused on 2 problems: identification of the signal that reaches the fibroblast from outside and tells it to grow and identification of metabolic reactions inside the cell that commit it to initiate DNA synthesis after the signal arrives. Although the signal and the metabolic reactions have not yet been clearly identified, and the relationship between in vivo conditions and the result of these in vitro studies still has to be determined, the large body of data collected so far and the steadily growing information concerning these problems suggest a complex interrelation between cellular environment and metabolic processes involved in growth regulation.

Density-dependent regulation of growth of BSC-1 cells in cell culture: growth inhibitors formed by the cells.

Inhibitors formed by a monkey epithelial cell line, BSC-1, play an important role in limiting growth at high cell densities. At least three inhibitors are formed: lactic acid, ammonia, and an unidentified inhibitor that may be an unstable protein. The unidentified inhibitor is destroyed by shaking the conditioned medium, by bubbling gas through the medium, or by heating or storing the medium in the absence of cells. The concentrations of lactic acid and ammonia that accumulate in conditioned medium inhibit growth when added to fresh medium. These results, together with earlier studies, indicate that density-dependent regulation of growth of BSC-1 cells results from the combined effects of (a) inhibitors formed by the cells, (b) decreased availability of receptor sites for serum growth factors as the cells become crowded, and (c) limiting concentrations of low molecular weight nutrients in the medium. In contrast, density-dependent regulation of growth in 3T3 mouse embryo fibroblasts results almost entirely from inactivation of serum factors.